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Fig. 4 NRF2 activation by curcumin normalizes inflammation in cftr-depleted zebrafish. (A) Control MO and cftr MO were pre-treated with DMSO (as control) or curcumin before injury, then injured and immediately put back in treatments until analysis. (B) mRNA levels of nrf2a at 2 hpA (relative gene expression from at least 3 independent experiments performed in triplicates, Two-way ANOVA with Tukey's multiple comparisons test). (C) Neutrophil number at the wound at 2 hpA in control MO and cftr MO TgBAC(mpx:EGFP)i114 (3 independent experiments, Two-way ANOVA with Tukey's multiple comparisons test). (D) Representative images of amputation-induced neutrophil mobilization in cftr-MO treated or not with curcumin (scale bars, 100 μm). (E) Inhibition of nrf2a was carried out in CF animals by injecting the nrf2a morpholino. Neutrophil number at the wound at 2 hpA (3 independent experiments, Two-way ANOVA with Tukey’s multiple comparisons test). (F) Schematic illustration of laser-mediated injury assay triggering ROS production by epithelial cells. (G-H) cftr MO are stained with CellROX®, treated, laser-injured, then ROS production was time-lapse imaged and quantified over 90 minutes by confocal microscopy. (G) ROS intensity at the wound site over the time course of inflammation. Line of best fit shown is calculated by linear regression. P-value shown is for the difference between the 2 slopes (n = 12, performed as 3 independent experiments). (H) Pseudocolored imaging of laser-injured caudal fin showing representative ROS production throughout inflammation. (I-M) mRNA levels of duox, nqo1, hmox1, cxcl8 and il1β at 2 hpA (duox, cxcl8 and il1β) or 8 hpA (nqo1 and hmox1) determined by RT qPCR (relative gene expression from at least 3 independent experiments performed in triplicates, Two-way ANOVA with Tukey's multiple comparisons test). (N-O) cftr MO Tg(il1b:eGFP-F)ump3 [19] were caudal fin amputated then the expression of Il1β was observed and analyzed. Relative Il1β intensity at 2hpA in cftr MO treated or not with curcumin (n = 18 from 3 independent experiments; Mann-Whitney U test) (N) and associated pseudocolored photomicrographs of injured tails (O) revealing il1β expression at the wound-edge (scale bar, 100 μm).